Targeting of cytokine antagonists

ABSTRACT

The present invention relates to a fusion protein, comprising a cytokine antagonist and a targeting moiety, preferably an antibody or anti-body like molecule. In a preferred embodiment, the cytokine antagonist is a modified cytokine which binds to the receptor, but doesn&#39;t induce the receptor signalling. The invention relates further to a fusion protein according to the invention for use in treatment of cancer and immune- or inflammation-related disorders.

The present invention relates to a fusion protein, comprising a cytokineantagonist and a targeting moiety, preferably an antibody or antibodylike molecule. In a preferred embodiment, the cytokine antagonist is amodified cytokine which binds to the receptor, but doesn't induce thereceptor signalling. The invention relates further to a fusion proteinaccording to the invention for use in treatment of cancer or for use intreatment of autoimmune diseases.

Cytokines are critical mediators of defence mechanisms against microbialinvasion and tumorigenesis. However, their production and activitiesmust be tightly regulated to prevent an excessive activity that canculminate in the uncontrolled inflammation and tissue injury, ascharacteristically observed with many autoimmune diseases.

Rheumatoid arthritis is the classic example of an autoimmune diseasewhere TNFα, IL-1, and IL-6 play a prominent role in the recruitment oflymphocytes and other types of leukocytes that mediate a progressivejoint destruction. TNF inhibitors have been shown to decrease symptoms,slow disease progression, and improve the quality of life for manypatients with rheumatoid arthritis (Moreland, 2009). Similarly, a mAbneutralizing IL-12 and IL-23 (ustekinumab) provides a potential therapyfor psoriasis (Elliott et al., 2009) and a recombinant human IL-1receptor antagonist, (anakinra, Kineret™), first approved by the FDA in2001 for the treatment of rheumatoid arthritis, is a promising agent forthe treatment of many IL-1-mediated autoinflammatory diseases(Goldbach-Mansky, 2009).

Several lines of evidence support the notion that overproduction of typeI interferon by plasmacytoid dendritic cells is the primary pathogenesisof several autoimmune diseases, including systemic lupus erythematosus,a multi-system autoimmune disease that affects skin, kidney,musculoskeletal, and hematologic tissues, and Sjogren's syndrome, adisease characterized by the destruction of glands producing tears andsaliva and which impacts 1-3% of the human population. Indeed, if thenatural IFN production is not regulated properly, the ensuing prolongedtype I IFN exposure can drive autoantibody production which promotes theonset of systemic autoimmune disease (Kiefer et al., 2012). Accordingly,novel therapeutics targeting type I IFN have been developed. Forinstance, two monoclonal antibodies which neutralize IFNα (Sifalimumaband Rontalizumab) are currently in clinical trials (McBride et al.,2012; Merrill et al., 2011) and a type I IFN antagonist has also beendesigned (Pan et al., 2008), (PCT/US2009/056366).

IL17A is the best characterized member of the IL17 family of cytokines.This pleiotropic cytokine interacts with a receptor composed of IL17RAand IL17RC subunits. The IL17RA chain is ubiquitously expressed,including haematopoietic, immune, epithelial, endothelial cell types, aswell as fibroblasts. IL17A is typically produced by Th17 cells uponactivation by a subset of cytokines including IL-1, IL-6, IL-21 andTGFβ, and propagates early inflammatory signals that serve to bridgeinnate and adaptive immune responses. IL17 is a potent activator ofneutrophils and plays an important role in the immune defence againstvarious extracellular pathogens. It is also well established that IL17Apromotes autoimmune pathologies (Geffen, 2009; Shen & Geffen, 2008).Brodalumab, Secukinumab and Ixekizumab target the IL17A/IL17R axis fortreatment of auto-immune diseases such as psoriasis and Crohn's disease.All may inflict adverse side effects including enhanced risk ofinfections (Hueber et al. 2012; Spuls & Hooft, 2012). Specific targetingof IL17A antagonists to selected cell types such as airway epithelium(asthma), astrocytes (multiple sclerosis), synoviocytes andmonocytes/macrophages (rheumatoid arthritis) or keratinocytes(psoriasis) may therefore offer a significant advantage over completelyantagonising IL17 function.

IL1α and ILβ are the founding members of the IL1 cytokine family. Bothare pleiotropic and function through a ubiquitously expressed receptorcomplex composed of IL-1 receptor type-I (IL-1RI) and IL-1 receptoraccessory protein (IL-1RAcP). Overactivation of this IL-1 axis isassociated with many human pathologies including rheumatoid arthritis(RA), chronic obstructive pulmonary disease (COPD), asthma, inflammatorybowel diseases, multiple sclerosis, atherosclerosis and Alzheimer'sdisease. Many immune cells of different lineages are activated by IL-1,including innate immune cells such as dendritic cells, macrophages andneutrophils, and also cells involved in the adaptive immune responseincluding naïve, Th17 and CD8+ T cells, and B cells (reviewed in Simsand Smith, 2010). Recombinant human IL-1RA (IL1 receptor antagonist, akaanakinra) can be used to treat rheumatoid arthritis and is beingevaluated for use in a wide spectrum of autoinflammatory diseases(Dinarello, 2011). One of the major side effects of prolonged treatmentwith anakinra is however the increased occurrence of infections.Selectively antagonising of IL-1 activity on only a subset of (immune)cells therefore may offer a safer alternative. It can be envisaged thattargeted inhibition of IL-1 action on selected innate immune cells,leaving its activity on the T cell compartment intact, may still showefficacy for the treatment of inflammatory diseases, without affectingthe host defence against pathogens.

Although the IL-7-related cytokine TSLP (thymic stromal lymphopoietin)is best studied in the context of promoting Th2 responses, it is nowclear that it functions on various immune and non-immune cell types(reviewed in Roan et al., 2012). Its receptor is composed of the IL-7Rα,which is shared with IL-7, and the widely expressed TSLPRα, also knownas CRLF2 (Pandey et al., 2000). TSLP promotes Th2-type inflammation byacting on several distinct cell types, including dendritic cells, CD4and CD8 T cells, B cells, NKT cells, mast cells, eosinophils andbasophils. It supports host defence against helminth parasites, but cancontribute to allergic inflammation, and antagonising TSLP was suggestedas a treatment for allergic diseases.

Conversely, TSLP can have a protective role in inflammatory diseasesdriven by exacerbated Th1 and Th17 responses, such as Inflammatory BowelDisease (reviewed in He and Geha, 2010 and Roan et al., 2012). It wasrecently also found that mutations in the TSLPRα are associated withcancer, including leukemias with poor prognosis (Harvey et al., 2010;Yoda et al., 2010; Ensor et al., 2011), and TSLP levels are correlatedwith breast cancer progression (Olkhanud et al., 2011) and reducedsurvival in pancreatic cancer (De Monte et al., 2011). Selectivetargeting of TSLP antagonists to selected tumor cell types therefore mayoffer a selective antitumor strategy, and additional modulation bytargeted antagonism of selected immune cells may be used to furtheroptimise such strategy. Similar approaches could also be undertaken fornon-malignant diseases.

The main problem with the therapeutic approaches aiming to neutralizecytokine actions is that the cytokine antagonists are not targetedtowards cells or tissues that are specifically involved in the onset ofthe autoimmune or autoinflammatory diseases. For example, It is easilyforeseeable that a long term systemic neutralization of type I IFNactivity by a monoclonal antibody or an IFN receptor antagonist carry animportant risk in term of viral infection susceptibility and tumordevelopment since type I IFN is a family of proteins essential in thecontrol of viral infections and for establishing immune responses,particularly those controlling cancer cell growth (Gajewski et al.,2012). Similarly, it is expected that a systemic neutralization of IL-1activity will impact the expansion, effector function, tissuelocalization, and memory response of antigen-cytotoxic T cells duringimmune responses (Ben-Sasson et al., 2013).

Surprisingly we found that specific targeting of the cytokine antagonistto a subset of target cells allows reaching the therapeutic effect,without having the negative side effects of systemic cytokine antagonistapplication. The invention is exemplified by targeting the action of atype I IFN antagonist to specific cell types expressing a given cellsurface marker. Such a method is applied to the design and constructionof a targeted IFN antagonist that inhibits the action of endogenous IFNspecifically on the cell subset culpably involved in the onset ofautoimmune diseases, leaving the other cells and organs fullyresponsive.

Although not yet approved, oncolytic viruses are advancing throughclinical trials (Russell et al., 2012). Oncolytic viruses are oftendesigned for having attenuated replication capacity in normal tissues byengineering their sensitivity to the normal cellular interferon-mediatedantiviral responses. An example is an oncolytic vesicular stomatitisvirus coding for interferon β (Naik et al., 2012). The therapeuticeffect of such viruses is expected to be a consequence of the defect ofthe IFN response exhibited by many tumor cells. However, the geneticheterogeneity of tumors that impact the IFN response is highly variableand impairs the efficacy of virus-mediated tumor lysis (Naik andRussell, 2009). Therefore, by inhibiting the IFN response specificallyin tumor cells, a tumor-targeted IFN antagonist would permit thespecific destruction of tumor cells by an oncolytic virus.

A first aspect of the invention is a fusion protein comprising acytokine antagonist and a targeting moiety consisting of an antibody oran antibody like molecule. A cytokine antagonist as used here can be anycytokine antagonist known to the person skilled in the art, includingbut not limited to a soluble receptor, a cytokine binding antibody or amutant cytokine. Preferably said cytokine antagonist is a mutantcytokine, even more preferably a mutant which binds to the receptor, butis not or only weakly inducing the cytokine signalling. Preferably, theaffinity of the mutant for the receptor is comparable to that of thewild type cytokine, even more preferable it has a higher affinity;preferably the signalling induced by the mutant is less than 20% of thatof the wild type, even more preferably less than 10% of that of the wildtype, even more preferably less than 5%, even more preferably less than1%. Most preferably, the binding of the mutant cytokine does not resultin detectable signalling. Such mutant can act as a competitive inhibitorof cytokine signalling. An antibody or antibody like molecule as usedhere is a protein specifically designed to bind another molecule,preferably a proteineous molecule, and comprising the specific bindingdomains. As a non-limiting example, said antibody or antibody likemolecule can be a heavy chain antibody (hcAb), single domain antibody(sdAb), minibody (Tramontano et al., 1994), the variable domain ofcamelid heavy chain antibody (VHH), the variable domain of the newantigen receptor (VNAR), affibody (Nygren et al., 2008), alphabody(WO2010066740), designed ankyrin-repeat domain (DARPins) (Stumpp et al.,2008), anticalin (Skerra et al., 2008), knottin (Kolmer et al., 2008)and engineered CH2 domain (nanoantibodies; Dimitrov, 2009). Thedefinition, as used here, excludes the Fc tail (without the bindingdomains) of an antibody. Preferably, said antibody or antibody likemolecule consists of a single polypeptide chain, even more preferably,said antibody is not post-translationally modified. Post-translationalmodification, as used here, indicates the modifications carried out byliving cell during or after the protein synthesis, but excludesmodifications, preferably chemical modifications, carried out on theisolated protein such as, but not limited to pegylation Even morepreferably said antibody or antibody-like molecule comprises thecomplementary determining regions, derived from an antibody. Mostpreferably, said targeting antibody or antibody-like molecule is ananobody.

Preferably, said cytokine antagonist and said targeting moiety areconnected by a linker, preferably a GGS linker. Preferably said GGSlinker contains at least 5 GGS repeats, more preferably at least 10 GGSrepeats, even more preferably at least 15 GGS repeats, most preferablyat least 20 GGS repeats.

In a preferred embodiment, the cytokine antagonist according to theinvention is an interferon antagonist; even more preferably, it is anIFNα2-R120E mutant. In another preferred embodiment, the cytokineantagonist according to the invention is an antagonist of a cytokine ofthe IL17 family, preferably an IL17A antagonist. In still anotherpreferred embodiment, the cytokine antagonist according to the inventionis an antagonist of the IL1 cytokine family, preferably an IL1α or ILβantagonist. In still another preferred embodiment, the cytokineantagonist according to the invention is a TSLP antagonist.

In one preferred embodiment, the antibody or antibody-like molecule isdirected against a cancer cell marker. Cancer cell markers are known tothe person skilled in the art, and include, but are not limited to CD19,CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, HER2,MUC1, GPNMB and PMSA. Preferably, said cancer marker is CD20 or HER2.

In another preferred embodiment, the antibody or antibody-like moleculeis directed against a marker on an immune cell, preferably aninflammatory cytokine producing immune cell. An immune cell, as usedhere, is a cell that belongs to the immune system, including but notlimited to monocytes, dendritic cells and T-cells. Preferably, saidimmune cell is a pro-inflammatory cytokine producing cell.

Markers of inflammatory cytokine producing cells are known to the personskilled in the art and include but are not limited to CD4, CD11b, CD26,sialoadhesin and flt3 receptor.

Another aspect of the invention is a fusion protein according to theinvention for use in treatment of cancer. Still another aspect of theinvention is a fusion protein according to the invention for use intreatment of autoimmune diseases.

Another aspect of the invention is a method to treat cancer, comprising(i) determination the type of cancer and the suitable targetingmarker(s) for the cancer cells in a patient suffering from cancer (ii)providing to said patient in need of the treatment a fusion proteincomprising a cytokine antagonist and a targeting moiety consisting of anantibody or an antibody-like molecule according to the invention,possibly with a suitable excipient. It is obvious for the person skilledin the art that the targeting moiety of step (ii) will be directed tothe targeting marker identified in step (i). Possible cancer cellmarkers are known to the person skilled in the art, and include, but arenot limited to CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74,CD138, AGS16, HER2, MUC1, GPNMB and PMSA.

Still another aspect of the invention is a method to treat an autoimmunedisease, comprising (i) determination in a patient suffering from anautoimmune disease the suitable targeting marker(s) for the immune cellscells (ii) providing to said patient in need of the treatment a fusionprotein comprising a cytokine antagonist and a targeting moietyconsisting of an antibody or an antibody-like molecule according to theinvention, possibly with a suitable excipient. Immune cells, as usedhere, include but are not limited to dendritic cells, CD4 and CD8 Tcells, B cells, NKT cells, mast cells, eosinophils and basophils.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Representation of the structural elements of thenanobody-hIFNα2-R120E fusion protein.

FIG. 2: Quantification of the luciferase activity induced by 10 pMhIFNα2 in the presence or absence (untreated) of the 4-11-hIFNα2-R120Efusion protein on HL116 (A) and HL116-mLR10 (B) cells.

FIG. 3: Quantification of the luciferase activity induced by 1 pM IFNβin the presence or absence (untreated) of the 4-11-hIFNα2-R120E fusionprotein on HL116 (A) and HL116-mLR10 (B) cells.

FIG. 4: FACS analysis of pY701-STAT1 in CD19 positive and negative humanPBMCs left untreated (left panel), treated with 50 pM of hIFNα2 (center)or with 50 pM of hIFNα2 in the presence of the CD20-targeted IFNantagonist.

FIG. 5: Density of the Daudi cell cultures treated by the followingcomponents:

A: Untreated

B: hIFNα2. 2 pM

C: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E. 1 μg/ml

D: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E. 0.1 μg/ml

E: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E-R149A. 3 μg/ml

F: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E-R149A. 1 μg/ml

G: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E-L153A. 3 μg/ml

H: hIFNα2. 2 pM+2HCD25-20×GGS-hIFNα2-R120E-L153A. 1 μg/ml

EXAMPLES Materials & Methods to the Examples Nanobody-IFN AntagonistFusion Construction

Using the QuikChange II-E Site-Directed Mutagenesis Kit (Agilent), themutation R120E which abrogates IFN-IFNAR1 binding and confers theantagonistic behaviour of human IFNα2 (Pan et al., 2008),(PCT/US2009/056366), was introduced into the pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNα2 construct (PCT/EP2013/050787), which isa fusion between a nanobody against the murine leptin receptor and thehuman IFNα2.

Production of the Nanobody-IFN Antagonist Fusion Protein

Hek 293T cells were transfected with the protein fusion constructs usingthe standard lipofectamin method (Invitrogen). 48 hours after thetransfection culture mediums were harvested and stored at −20° C.

Cell Lines

Hek 293T cells were grown in DMEM supplemented with 10% FCS. The HL116clone (Uze et al., 1994) is derived from the human HT1080 cell line. Itcontains the firefly luciferase gene controlled by the IFN-inducible6-16 promoter. The derived HL116-mLR10 clone which expresses the murineleptin receptor was described (PCT/EP2013/050787).

Measurement of the Luciferase Activities

Antagonistic IFN activities were measured by quantifying the inhibitionof the luciferase activity induced in HL116 cells and on the HL116-mLR10expressing the mLR by IFNα2 or IFNβ. The IC50 values were calculatedusing nonlinear data regression with Prism software (GraphPad).Luciferase activities were determined on a Berthold Centro LB960luminometer using a luciferase substrate buffer (20 mM Tricine, 1.07 mM(MgCO3)4Mg(OH)2•5H20, 2.67 mM MgSO4•7H2O, 0.1 mM EDTA, 33.3 mMdithiothreitol, 270 μM coenzyme A, 470 μM luciferin, 530 μM ATP, finalpH 7.8) after 6 hr IFN stimulation.

Example 1: The Nanobody-IFNα2-R120E Fusion Protein

The nanobody 4-11, directed against the murine leptin receptor was fusedto the IFNα2 mutant R120E as described in the materials and methods

FIG. 1 shows a schematic representation of the nanobody-IFN antagonistfusion protein constructed with the nanobody 4-11 against the murineleptin receptor and the human IFNα2-R120E (numbering as in Piehler etal., 2000).

Example 2: Targeted Inhibition of IFNα Activity on mLR-Expressing Cells

Parental HL116 cells and the derived HL116-mLR10 cells which express themouse leptin receptor were treated for 6 hours with 10 pM IFNα2 in thepresence of several dilutions of culture medium conditioned by Hek 293Tcells expressing the 4-11-IFNα2-R120E fusion protein. The 10 pM IFNα2dose was chosen because it corresponds to the IFNα2 EC50 on both celllines. Cells were then lysed and the IFN-induced luciferase activity wasquantified. At the higher concentration tested, the 4-11-IFNα2-R120Efusion protein was unable to inhibit IFNα2 action on untargeted HL116cells (FIG. 2A). In contrast, its dose-dependent inhibition effect isclear on HL116-mLR10 cells which express the target of the 4-11 nanobody(FIG. 2B).

Example 3: Targeted Inhibition of IFNβ Activity on mLR-Expressing Cells

Among the subtypes which constitute the human type I IFN, the IFNβ showsthe highest affinity for the IFNα/β receptor. We thus tested whether the4-11-IFNα2-R120E fusion protein exerts also an antagonistic activityagainst IFNβ action.

Parental HL116 cells and the derived HL116-mLR10 cells which express themouse leptin receptor were treated for 6 hours with 1 pM IFNβ in thepresence of several dilutions of culture medium conditioned by Hek 293Tcells expressing the 4-11-IFNα2-R120E fusion protein. The 1 pM IFNβ dosewas chosen because it corresponds to the IFNβ EC50 on both cell lines.Cells were then lysed and the IFN-induced luciferase activity wasquantified. At the higher concentration tested, the 4-11-IFNα2-R120Efusion protein was unable to inhibit IFNα2 action on untargeted HL116cells (FIG. 3A). In contrast, its dose-dependent inhibition effect isclear on HL116-mLR10 cells which express the target of the 4-11 nanobody(FIG. 3B).

Example 4. Specific Inhibition of IFNα2-Induced STAT1 Phosphorylation inB-Cells Within Human Whole PBMCs

The type I IFN antagonist IFNα2-R120E was fused to the anti-human CD20nanobody 2HCD25 through a linker sequence made with 20 repeats of GGSmotif. The fusion protein was produced in E. coli and purified byImmobilized Metal Affinity chromatography (IMAC).

Human peripheral blood mononuclear cells (PBMCs) are expected to contain≈4% of B-cells which can be characterized by the cell surface expressionof CD19. The large majority of circulating B-cells are also positive forthe expression of CD20.

PBMCs were isolated over ficoll gradient (histopaque-1077,Sigma-Aldrich) from blood samples of healthy donors. Cells were leftuntreated or were incubated for 15 minutes with 50 pM of human IFNα2 inthe absence or presence of 10 μg/ml of the 2HCD25 nanobody-IFNα2-R120Efusion protein.

Cells were then fixed (BD Fix Buffer I), permeabilized (BD Perm BufferIII) and labelled with PE-labelled anti pSTAT1 (BD#612564) andAPC-labelled anti human CD19 (BD#555415). FACS data were acquired usinga BD FACS Canto and analyzed using Diva (BD Biosciences) software forthe fluorescence associated with pSTAT1 in CD19 positive and negativecell populations.

FIG. 4 shows that the IFN antagonist linked to the nanobody specific forCD20 inhibits the IFN action specifically in the major part of the Bcell population, leaving intact the IFN response in the CD19 negativecell population.

Example 5. The CD20-Targeted Type I IFN Antagonist Inhibits theAntiproliferative Activity of type I IFN

Having established that the fusion protein of the 2HCD25 nanobody andIFNα2-R120E inhibits IFN-induced STAT1 phosphorylation specifically inB-cells, we tested if it can inhibit the antiproliferative activity oftype I IFN. In addition, we evaluated the effect of the IFN mutationsL153A and R149A that decrease the affinity of IFNα2 for IFNAR2 by afactor of 10 and 100, respectively, in combination with the inhibitingmutation R120E.

Daudi cells are a human lymphoblastoid B-cell line expressing CD20.Daudi cells were seeded at 2.0×105 cells/ml and were left untreated orcultured for 72 h in the presence of 2 pM IFNα2 alone or in combinationwith various CD20-targeted IFN antagonists. They were then counted toestimate the efficacy of the inhibition of proliferation induced byIFNα2. FIG. 5 shows that the CD20-targeted IFN antagonist fully inhibitsthe antiproliferative activity of IFNα2. It also shows that decreasingthe IFN-IFNAR2 affinity decreases the antagonistic activity, provingthat the inhibitory effect is indeed due to the binding of the targetedantagonist.

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1.-13. (canceled)
 14. A method for treating an autoimmune diseasecomprising providing to a subject in need thereof an effective amount ofa composition comprising a fusion protein, the fusion protein comprisingan interferon antagonist and a variable domain of camelid heavy chainantibody (VHH) or a variable domain of new antigen receptor (VNAR),wherein the interferon antagonist is a human IFNα2 comprising an R120Emutation and wherein the VHH or VNAR is directed to CD20.
 15. The methodof claim 14, wherein the fusion protein further comprises a linkerconnecting the interferon antagonist and the VHH or VNAR.
 16. The methodof claim 14, wherein the human IFNα2 further comprises a second mutationthat decreases binding activity of the interferon antagonist.
 17. Themethod of claim 16, wherein the second mutation is R149A.
 18. The methodof claim 17, wherein the fusion protein further comprises a linkerconnecting the interferon antagonist and the VHH or VNAR.